Retention of enzyme activity with a boron-doped diamond electrode in the electro-oxidative nitration of lysozyme

J Iniesta, M Deseada Esclapez-Vicente, J Heptinstall, DJ Walton, IR Peterson, VA Mikhailov, Helen Cooper

Research output: Contribution to journalArticle

5 Citations (Scopus)

Abstract

In this paper we report the successful use of a non-metallic electrode material, boron-doped diamond (BOO), for the anodic electro-oxidative modification of hen egg white lysozyme (HEWL). Platinum electrodes can give rise to loss of activity of HEWL in elect rosynthetic studies, whereas activity is retained on boron-doped diamond which is proposed as an effective substitute material for this purpose. We also compare literature methods of electrode pre-treatment to determine the most effective in electrosynthesis. Our findings show a decrease in total nitroprotein yield with decreasing nitrite concentration and an increase with increasing solution pH, confirming that, at a BDD electrode, the controlling factor remains the concentration of tyrosine phenolate anion. Purification of mono- and bis-nitrated HEWL and assay of enzymic activity showed better retention of activity at BOO electrode surfaces when compared to platinum. The products from electro-oxidation of HEWL at BOO were confirmed by electrospray ionization Fourier transform ion cyclotron resonance (ESI-FT-ICR) mass spectrometry, which revealed unique mass increases of +45 and +90 Da for the mono- and bis-nitrated lysozyme, respectively, corresponding to nitration at tyrosine residues. The nitration sites were confirmed as Tyr23 and Tyr20. Crown Copyright (C) 2010 Published by Elsevier Inc. All rights reserved.
Original languageEnglish
Pages (from-to)472-478
Number of pages7
JournalEnzyme and Microbial Technology
Volume46
Issue number6
DOIs
Publication statusPublished - 1 May 2010

Keywords

  • Electro-oxidative nitration
  • Lysozyme
  • Nitrotyrosine
  • BDD
  • Infrared multiphoton dissociation
  • Tandem mass spectrometry

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